High oil prices and growing concerns over national security and climate change are driving investment and innovation in the renewable biofuels sector [Wirth et al., 2003; Kerr and Service, 2005; Hill et al., 2006]. Unlike fossil fuels—such as coal, petroleum, and natural gas, which are finite resources—biofuels are a renewable source of energy that can be replenished on an ongoing basis. Further, because biofuels are generally derived from plants, which absorb carbon from the atmosphere as they grow, biofuel production offers the potential to help of set carbon dioxide (CO2) emissions and mitigate climate change [Antoni et al., 2007]. Photosynthetic algae and cyanobacteria have been proposed for producing biofuels through a direct photoconversion process [Atsumi et al, 2009]. The advantage of photosynthetic microbes is that they grow over a range of temperatures, pH and nutrient conditions, and can be cultivated in large scale in ponds or closed photobioreactors. So far, more than 40,000 species of photosynthetic microbes have been identified, with the expectation that many more will be discovered. Their potential application for biofuels production has not yet been fully evaluated. In addition, synthetic biology tools have been recently used to modify the photosynthetic microbes to generate various novel high energy-content biofuels directly from sunlight and carbon dioxide [Atsumi et al. 2009]. The efficiency of the biofuel production depends on the photosynthetic activity of microbes, e.g. the ability of consumption of CO and the generation of oxygen (OC) [Angermayr et al. 2009].
Several methods have been developed to measure photosynthetic activities [Millan-Almaraz et al., 2009]. Typically, these methods involve measuring a single parameter, either O2 generation or CO2 consumption. These methods include: a) dry matter accumulation; b) manometric measurement of the pressure of CO2 or O2 in an isolated chamber containing photosynthetic organisms; c) use of electrodes to measure dissolved oxygen and CO2 or change in pH; d) CO2 and/or O2 gas exchange; e) CO2 isotope measurement; and f) measurement of autofluorescence from chlorophyll and/or chloroplast [Millan-Almaraz et al., 2009]. Although these methods have been applied successfully in past research, they are typically time- and labor-intensive, they often require special devices, and their measurement throughput is typically low. Among them, measurements of the CO2 consumption and/or O2 generation using electrodes [Clark, 1956] are currently the most popular technique.
Fluorescence-based optical sensors, in which organic and polymeric fluorophores are deposited onto different surfaces, can be miniaturized easily to sub-micrometer scale, and have been applied to measure pH and O2 changes in both small and large dimension scales [Nagl and Wolfbeis, 2007; Amao, 2003]. These methods have been demonstrated to be sensitive and highly reproducible, and can readily be developed into high throughput formats.
One problem in developing optical sensors for measuring pH and dissolved oxygen in photosynthetic organisms is that the sensor must possess stronger fluorescence intensities than that of the organism itself. Due to their photosynthetic activity, green algae and cyanobacteria contain significant amounts of chlorophyll, nicotinamide adenine dinucleotide phosphate (NADPH), and other pigments that exhibit strong autofluorescence under light excitation [Kühl, 2005; Steigenberger et al., 2004; Mi et al., 2000]. Thus, the optical sensor must be able to alleviate the background interference caused by chlorophyll and other pigments.